Systems and methods for monitoring the amplification of dna

ABSTRACT

A system and method for amplifying and detecting nucleic acids are disclosed. In one embodiment, the system includes: a microfluidic device comprising a channel for receiving a sample of solution containing real-time PCR reagents; a temperature control system configured to cycle the temperature of the sample; an excitation source for illuminating the sample; a fiber optic probe comprising (i) an optical fiber having a distal end and a proximal end and (ii) a probe head connected to the distal end of the optical fiber and positioned between the distal end of the optical fiber and the channel; and a detector configured to detect emissions exiting the proximal end of the optical fiber.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.14/697,036, filed on Apr. 27, 2015, which is a divisional of U.S. patentapplication Ser. No. 12/144,223, filed on Jun. 23, 2008, now U.S. Pat.No. 9,017,946, the disclosures of each of which are hereby incorporatedby reference in their entireties.

BACKGROUND Field of the Invention

This invention pertains to systems and methods for amplifying anddetecting nucleic acids. In one embodiment, it pertains to methods formonitoring a polymerase chain reaction (PCR) in a microfluidic system.

Discussion of the Related Art

The detection of nucleic acids is central to medicine, forensic science,industrial processing, crop and animal breeding, and many other fields.The ability to detect disease conditions (e.g., cancer), infectiousorganisms (e.g., HIV), genetic lineage, genetic markers, and the like,is ubiquitous technology for disease diagnosis and prognosis, markerassisted selection, correct identification of crime scene features, theability to propagate industrial organisms and many other techniques.Determination of the integrity of a nucleic acid of interest can berelevant to the pathology of an infection or cancer. One of the mostpowerful and basic technologies to detect small quantities of nucleicacids is to replicate some or all of a nucleic acid sequence many times,and then analyze the amplification products. PCR is perhaps the mostwell-known of a number of different amplification techniques.

PCR is a powerful technique for amplifying short sections of DNA. WithPCR, one can quickly produce millions of copies of DNA starting from asingle template DNA molecule. PCR includes a three phase temperaturecycle of denaturation of DNA into single strands, annealing of primersto the denatured strands, and extension of the primers by a thermostableDNA polymerase enzyme. This cycle is repeated so that there are enoughcopies to be detected and analyzed. In principle, each cycle of PCRcould double the number of copies. In practice, the multiplicationachieved after each cycle is always less than 2. Furthermore, as PCRcycling continues, the buildup of amplified DNA products eventuallyceases as the concentrations of required reactants diminish. For generaldetails concerning PCR, see Sambrook and Russell, Molecular Cloning—ALaboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology,F. M. Ausubel et al., eds., Current Protocols, a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(supplemented through 2005) and PCR Protocols A Guide to Methods andApplications, M. A. Innis et al., eds., Academic Press Inc. San Diego,Calif. (1990).

Real-time PCR refers to a growing set of techniques in which onemeasures the buildup of amplified DNA products as the reactionprogresses, typically once per PCR cycle. Monitoring the accumulation ofproducts over time allows one to determine the efficiency of thereaction, as well as to estimate the initial concentration of DNAtemplate molecules. For general details concerning real-time PCR seeReal-Time PCR: An Essential Guide, K. Edwards et al., eds., HorizonBioscience, Norwich, U.K. (2004).

Several different real-time detection chemistries now exist to indicatethe presence of amplified DNA. Most of these depend upon fluorescenceindicators that change properties as a result of the PCR process. Amongthese detection chemistries are DNA binding dyes (such as SYBR® Green)that increase fluorescence efficiency upon binding to double strandedDNA. Other real-time detection chemistries utilize Foerster resonanceenergy transfer (FRET), a phenomenon by which the fluorescenceefficiency of a dye is strongly dependent on its proximity to anotherlight absorbing moiety or quencher. These dyes and quenchers aretypically attached to a DNA sequence-specific probe or primer. Among theFRET-based detection chemistries are hydrolysis probes and conformationprobes. Hydrolysis probes (such as the TaqMan® probe) use the polymeraseenzyme to cleave a reporter dye molecule from a quencher dye moleculeattached to an oligonucleotide probe. Conformation probes (such asmolecular beacons) utilize a dye attached to an oligonucleotide, whosefluorescence emission changes upon the conformational change of theoligonucleotide hybridizing to the target DNA.

A number of commercial instruments exist that perform real-time PCR.Examples of available instruments include the Applied Biosystems PRISM7500, the Bio-Rad iCylcer, and the Roche Diagnostics LightCycler 2.0.The sample containers for these instruments are closed tubes whichtypically require at least a 10 μl volume of sample solution. If thelowest concentrations of template DNA detectable by a particular assaywere on the order of one molecule per microliter, the detection limitfor available instruments would be on the order of tens of targets persample tube.

More recently, a number of high throughput approaches to performing PCRand other amplification reactions have been developed, e.g., involvingamplification reactions in microfluidic devices, as well as methods fordetecting and analyzing amplified nucleic acids in or on the devices.Thermal cycling of the sample for amplification is usually accomplishedin one of two methods. In the first method, the sample solution isloaded into the device and the temperature is cycled in time, much likea conventional PCR instrument. In the second method, the sample solutionis pumped continuously through spatially varying temperature zones.

U.S. patent application Ser. No. 11/505,358, entitled, “Real-time PCR inmicro-channels,” which is assigned to the assignee of this applicationand which is incorporated herein by this reference in its entirety,describes, among other things, a novel method to acquire real-time PCRdata in a microfluidic system. One of the steps in that method is tocapture an image of a fluorescent signal along the length of at leastone microfluidic channel.

A conventional apparatus to capture an image of a fluorescent signal isillustrated in FIG. 1. As illustrated in FIG. 1, the light emitted fromthe material under study is collected by a high numerical apertureobjective and the light is re-imaged onto a two-dimensional detectorarray.

A reason for using a high numerical aperture objective to collectluminescence is that the solid angle subtended is higher, and thereforethe photon collection efficiency is higher, than that achieved using alow numerical aperture objective. In certain cases, collectionefficiency may be an important parameter because, in certain cases,emitted light flux is often so low that signal levels at the detectorare weak. Therefore, at least in certain cases, it is desirable tomaximize collection efficiency.

The drawback of using a high numerical aperture microscope objective isthat the imaged area is small. The effective field of view of aconventional fluorescence microscope imaging system might have a lineardimension of 1 mm or smaller. This becomes a problem when the region ofinterest on a microfluidic chip is larger (e.g., if the length and widthare in the range of 10-100 mm).

One strategy to address the problem of imaging a large region ofinterest with high collection efficiency is to use an optical systemwith large diameter optics. This strategy has a benefit that most or allof the region of interest may be imaged simultaneously. An example ofthis approach is illustrated in U.S. Patent Application 2006/0006067,entitled, “Optical Lens System and Method for Microfluidic Devices,”which describes a multi-element lens system.

Another strategy would be to translate the sample holder with respect tothe optical system or vice versa (e.g. in a raster pattern) to collectpixel data in series. An example of this approach is described in U.S.Pat. No. 5,631,734, entitled, “Method and Apparatus for Detection ofFluorescently Labeled Materials.” This patent describes a system forcollecting fluorescence data from a substrate, for example a DNAmicroarray, in which the substrate is held by an x-y-z translation stageand translated in front of a microscope objective.

PCT publication WO 2005/075683 A1, entitled, “High Throughput Device forPerforming Continuous-Flow Reactions,” describes a continuous-flow PCRdevice that uses a fused silica capillary wrapped into a helix aroundthree temperature-controlled blocks. This publication shows a microscopeobjective lens being scanned transverse to the windings. Although thedescription is short on detail, presumably an entire optical imagingsystem, including lenses, beam-splitters, filters, and detectors, wouldhave to be scanned along as well.

U.S. Pat. No. 5,928,907, entitled, “System for Real Time Detection ofNucleic Acid Amplification Products,” describes a system for real-timePCR monitoring that uses a fiber optic and a lens to capturefluorescence from a closed, Eppendorf-style sample tube. The sample tubevolume was 200 ul, and the fiber optic and 8 mm diameter collection lenswere fixed with respect to the tube, looking down through the top of thetube and the airspace over the sample solution.

U.S. Patent Application 2005/0069257 A1, “Fiber Lens with MultimodePigtail” gives an example of a miniature lens system that is permanentlyaffixed to the end of an optical fiber. Further examples of miniaturefiber coupling systems can be found in product literature by CorningInc. for lensed fibers, tapered fibers, and gradient index fibers andlenses. These devices are used typically in telecommunication equipment,for example, for coupling light from a semiconductor diode laser into anoptical fiber, or for coupling light from one fiber into another fiber.

SUMMARY OF THE INVENTION

The present invention provides, among other things, improved systems andmethods for capturing an image of a fluorescent signal. In addition, thepresent invention may be useful in a variety of additional applications.

A system according to an embodiment of the invention includes: amicrofluidic device comprising a channel for receiving a sample ofsolution containing real-time PCR reagents; a temperature control systemconfigured to cycle the temperature of the sample; and an imaging systemfor detecting emissions from the sample, wherein the imaging systemcomprises: an excitation source for illuminating the sample, a fiberoptic probe comprising (i) an optical fiber having a distal end and aproximal end and (ii) a probe head connected to the distal end of theoptical fiber and positioned between the distal end of the optical fiberand the channel, and a detector configured to detect emissions exitingthe proximal end of the optical fiber.

The probe head had may be positioned directly above the channel and maybe positioned no more than about 10 millimeters from the top of thechannel. The system may also include: a positioning system configured toscan the fiber optic probe over at least a portion of the channel and apump for causing the sample to flow through the channel. In suchembodiments, the positioning system may be configured to scan the fiberoptic probe over the portion of the channel at a speed that is greaterthan the speed at which the sample is expected to flow through thechannel.

In some embodiments, the imaging system may also include a second fiberoptic probe comprising (i) an optical fiber having a distal end and aproximal end and (ii) a probe head connected to the distal end of theoptical fiber and positioned between the distal end of the optical fiberand the channel. In such a system, the first fiber optic probe may havea first field of view and the second fiber optic probe may have a secondfield of view, wherein a first portion of the channel is within thefirst field of view but not the second field of view and a secondportion of the channel is within the second field of view but not thefirst field of view.

With respect to the probe head, in some embodiments, the probe headincludes a ball lens, a gradient index lens, a liquid lens, or a highindex liquid. The probe head may have a diameter between about 0.1millimeters (mm) and 5 mm, but more preferably between about 0.5 mm and2 mm. Additionally, in some embodiments the distance between the probehead and the channel is less than 10 mm (e.g., about 1 mm in someembodiments). With respect to the optical fiber, in some embodiments,the optical fiber includes: a multimode fiber, a liquid filled fiber, aphotonic crystal fiber.

In some embodiments, the excitation source is optically connected to theoptical fiber such that when the excitation source emits excitationlight, the excitation light enters the optical fiber and then exits theoptical fiber through the distal end of the optical fiber.

In some embodiments, the microfluidic device further includes a secondchannel for receiving a second sample of solution containing real-timePCR reagents, and the imaging system further comprises a second fiberoptic probe comprising (i) an optical fiber having a distal end and aproximal end and (ii) a probe head connected to the distal end of theoptical fiber and positioned between the distal end of the optical fiberand the second channel, and a second detector configured to detectemissions exiting the proximal end of the optical fiber of the secondfiber optic probe. In such embodiments, the system may include apositioning system configured to scan the first fiber optic probe overat least a portion of the first channel and simultaneously scan thesecond fiber optic probe over at least a portion of the second channel.

In some embodiments, one or more filters may be positioned between theproximal end of the optical fiber and the detector. The one or morefilters may include a tunable wavelength filter.

A system according to another embodiment includes: a sample containerfor containing a sample of a solution containing real-time PCR reagents;a temperature control system configured to cycle the temperature of thesample; an excitation source for illuminating the sample; a fiber opticprobe comprising: a bundle of optical fibers including a central opticalfiber surrounded by a plurality of outer optical fibers, and a probehead connected to a distal end of the central optical fiber andpositioned between the distal end of the central optical fiber and thesample container, and a detector configured to detect emissions exitingthe proximal end of the central optical fiber, wherein the excitationsource is optically connected to each of the plurality of outer opticalfibers such that when the excitation source emits excitation light, theexcitation light enters the outer optical fibers and then exits theouter optical fibers through a distal end of the optical fibers. Thesample container may be in the form of a channel.

In some embodiments, the excitation source comprises at least two lightemitting devices, wherein each of the at least two light emittingdevices is optically connected to at lest one of the outer opticalfibers.

A method according to some embodiments of the invention includes: (a)moving a sample of test solution containing real-time PCR reagentsthrough a channel; (b) while the sample is moving through a section ofthe channel (i) cycling the temperature of the sample in order toachieve PCR, (ii) illuminating the sample with excitation light, and(iii) using a fiber optic probe to capture fluorescent light emittedfrom the sample; and (c) measuring the intensity of the fluorescentlight.

In some embodiments, the fiber optic probe includes: (i) an opticalfiber having a proximal end and a distal end and (ii) a probe headconnected to and positioned adjacent to the distal end of the opticalfiber, the probe head having a field of view and being positioned suchthat at least a portion of the section of the channel is within thefield of view. The probe head may be positioned directly above thechannel and may be positioned no more than about 10 millimeters from thetop of the channel.

In some embodiments, the method further includes moving the fiber opticprobe along at least the section of the channel while using the fiberoptic probe to capture the fluorescent light. The speed at which thefiber optic probe is moved along the section of the channel may begreater than the speed at which the sample moves through the channel (insome embodiments it may be at least 10 times greater).

In some embodiments, the method further includes using a second fiberoptic probe to capture fluorescent light emitted from the sample whilethe sample is moving through the section of the channel. In someembodiments, all the while the sample is moving through the section ofthe channel, the first fiber optic probe and the second fiber opticprobe are fixed in position relative to the channel.

The above and other embodiments of the present invention are describedbelow with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIG. 1 depicts a prior art apparatus to capture an image of afluorescent signal.

FIG. 2 is a block diagram illustrating a system according to someembodiments of the invention.

FIG. 3 is a diagram of a close-up side view of a possible probe headassembly.

FIG. 4 illustrates an embodiment of the present invention whereinexcitation light and fluorescence both travel along the same opticalfiber.

FIG. 5 illustrates an embodiment of the present invention whereinexcitation light and fluorescence both travel along the differentoptical fibers in a bundle of optical fibers.

FIG. 6 illustrates an embodiment of the present invention wherein theexcitation light is directed on to the sample without going through anoptical fiber.

FIG. 7A depicts a cross-sectional view of a version of the presentinvention utilizing a single probe.

FIG. 7B depicts a cross-sectional view of a version of the presentinvention utilizing multiple probes.

FIGS. 8A-8D illustrate a few of many possible trajectories probes maytake to scan an area of interest.

FIG. 9 depicts a bundle of optical fibers.

FIG. 10 depicts a positioning system coupled to a probe.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, the words “a” and “an” mean “one or more.”

Aspects of the present invention provide a system for detectingfluorescence emitted from a microfluidic device using at least one fiberoptic probe.

FIG. 2 illustrates a functional block diagram of a system 200 accordingto some embodiments of the invention. As illustrated in FIG. 2, system200 may include a microfluidic device 202. Microfluidic device 202 maybe a microfluidic chip. Microfluidic device 202 may include one or moremicrofluidic channels 204. In the example shown, device 202 includes twomicrofluidic channels, channel 204 a and channel 204 b. Although onlytwo channels are shown in the exemplary embodiment, it is contemplatedthat device 202 may have fewer than two or more than two channels. Forexample, in some embodiments, device 202 includes eight channels 204.

Device 202 may include two DNA processing zones, a DNA amplificationzone 231 (a.k.a., PCR zone 231) and a DNA melting zone 232. A DNA sampletraveling through the PCR zone 231 may undergo PCR, and a DNA samplepassing through melt zone 232 may undergo high resolution thermalmelting. As illustrated in FIG. 2, PCR zone 231 includes a first portionof channels 204 and melt zone 232 includes a second portion of channels204, which is down stream from the first portion.

In order to achieve PCR for a DNA sample flowing through the PCR zone231, the temperature of the sample must be cycled, as is well known inthe art. Accordingly, in some embodiments, system 200 includes atemperature control system 220. The temperature control system 220 mayinclude a temperature sensor, a heater/cooler, and a temperaturecontroller. In some embodiments, a temperature control system 220 isinterfaced with main controller 230 so that main controller 230 cancontrol the temperature of the samples flowing through the PCR zone andthe melting zone.

To monitor the PCR process and the thermal melting process that occur inPCR zone 231 and melt zone 232, respectively, system 200 may include animaging system 218. Imaging system 218 may include an excitation source253, a detector 250, a controller 251, and an image storage unit 252.

Further features of system 200 are described in U.S. patent applicationSer. No. 11/770,869, which is incorporated herein by this reference inits entirety.

Referring now to FIG. 3, an embodiment of imaging system 218 isillustrated. As shown in FIG. 3, imaging system 218 may include a fiberoptic probe 301 that includes a probe head 302 connected to an opticalfiber 306, which directs fluorescent light to a light sensor or detectorarray 250. Suitable detectors would include, but not be limited to:photomultiplier tubes; micro-channel plate detectors; photoconductors;photodiodes (include avalanche photodiodes); and detector arrasincluding CCD and CMOS detector arrays. Fixed and/or tunable wavelengthfilters 308 discriminate against unwanted wavelengths such as scatteredexcitation light. In addition, the fluorescence may be dispersedspectrally onto a plurality of detectors by using devices such asdiffraction gratings, prisms, or multilayer dielectric wavelengthfilters.

Excitation light may be directed onto the microfluidic device 202 in thesame location where the probe head 302 is set to collect emitted light.The excitation light may comprise light of multiple wavelengths and maybe generated by a variety of light sources. In addition, excitationlight may be directed onto the microfluidic device 202 in a variety ofways. In one embodiment, the excitation light source 253 is coupled tothe same fiber 306 used to carry captured fluorescence with couplingoptics 316. This embodiment may use, for example, a dichromatic filter314 to direct excitation light through coupling optics 316 and into theoptical fiber 306 on substantially the same path as the fluorescence,but in the opposite direction.

Referring now to FIG. 4, a diagram of a close-up side view of onepossible probe head 302 is shown. In general, when light is emitted fromthe microfluidic channel 204 a of microfluidic chip 202, it followslight path 412 and through probe head 302 is collected into the opticalfiber 306. It would be understood by one of ordinary skill in the artthat optical fiber 306 may comprise a single optical fiber or, as shownin FIGS. 9 & 10, a bundle of optical fibers.

As shown in FIG. 4, probe head may include a light collecting element408 connected to the distal end 402 of each optical fiber 306. Lightcollecting element 408 may comprise one or more of a high-indexspherical lens, gradient index lens, a Fresnel lens, a micro-lenssystem, a lensed fiber, or any combination thereof. Probe head 302 mayfurther comprise a spacer 410 positioned between the end 402 of thefiber 306 and light collecting element 408. Preferably, probe head 302is integrally connected to the optical fiber 306.

Probe head 302 is designed to capture a significant fraction of thelight emitted from within a channel of the microfluidic device 202. Bypositioning the probe head 302 close to the outer surface of the device,it is possible to achieve reasonably high collection efficiency with arelatively small diameter collecting element 408. In one embodiment,desirable collection efficiencies can be achieved by positioning theprobe head about 20 millimeters, and preferably about 10 millimeters,from the top of a channel of the microfluidic device 202. Of course,other distances between the probe head and the top of the channel may beused as well.

A scanner 490 can be connected to the probe head to scan the probe headacross an area of interest. Scanner 490 may include a positioner (e.g.,the MX80 positioner available from Parker Hannifin Corporation of PA(“Parker”)) for positioning probe head 302, a stepping drive (e.g., theE-AC Microstepping Drive available from Parker) for driving thepositioner, and a controller (e.g., the 6K4 controller available fromParker) for controlling the stepping drive.

Referring now to FIG. 5, another embodiment of imaging system 218 isillustrated. In the embodiment shown, the excitation light can becarried by at least one separate optical fiber. As shown in FIG. 5,light from the excitation source or sources 253 is directed through thecoupling optics 516 e to excitation optical fiber 506 e. Similarly,fluorescence from the probe head 302 is directed from fluorescenceoptical fiber 506 f through the coupling optics 516 f to filters 308 andthe detector or detectors 250. Fluorescence optical fiber 506 f andexcitation optical fiber 506 e can be bundled together to form opticalbundle 506 b.

Referring now to FIG. 6, another embodiment of imaging system 218 isillustrated. In the embodiment shown in FIG. 6, the excitation light isnot carried by a fiber, but is directed into the micro-channel throughfree space. As shown in FIG. 6, light from the excitation light sourceor sources 253 is directed on to the microfluidic chip 202 using mirror620. Mirror 620 may be movable so as to be capable of directing theexcitation light on to any desired point on the microfluidic chip 202.

Referring now to FIGS. 7A and 7B, a comparison of a single fiber opticprobe to a multiple fiber optic probe configuration is shown. A singleprobe 702 may be connected to a scanner that scans the probe over anarea of interest from different locations in series. The single probe702 can be scanned in one or two dimensions across the face of themicrofluidic chip 710.

Alternatively, in the embodiment shown in FIG. 7B, a plurality of fiberoptic probes 704 may be fixed with respect to the microfludic chip 710,and each collects fluorescence signal data from one location. As analternative to being fixed, the plurality of probe heads 704 could beconfigured so that they can be scanned over an area of interest and eachprobe head 704 can be used to gather image data from a section of thetotal area of interest.

An advantage of using multiple probes at the same time is that use ofmultiple probes creates some degree of parallelism and could be used todecrease the time required to collect the desired image data. This isespecially true if the probe diameter is comparable to, or smaller thanthe required spatial resolution in a particular direction.

Depending on a combination of parameters (e.g. probe head size, requiredspatial resolution, required signal acquisition durations, etc.), anumber of possible acquisition sequences and scanning trajectories arepossible. FIGS. 8A-8D illustrate a few of the possible scanningtrajectories. As shown in FIG. 8A, a 2-D scanner trajectory 802 could beused with a single probe. In this trajectory, the probe is moved over anarea of interest in two dimensions. As shown in FIG. 8B, by using moreprobes (e.g. five) an “almost” 1-D scanner trajectory 804 may be adoptedwherein several are scanned in one direction and then back again. Thetrajectory of FIG. 8C uses even more probes. A 1-D scanner trajectory806 can be used wherein the several probes are only moved across thearea of interest in one direction. FIG. 8D shows another trajectoryoption: a fixed array 808 of probes.

Referring now to FIGS. 9 and 10, another embodiment of probe head 302 isillustrated. Probe head 302 can comprise perimeter probe heads 902 andone or more inner probe heads 904. Each probe head 902 and 904 can beconnected to a different optical fiber 306 or 1002 (as shown in FIG.10). Optical fibers 306 can direct excitation light through perimeterprobe heads 902 and optical fiber 1002 can direct fluorescence collectedby inner probe head to a light detector. Each perimeter probe head 904may emit the same frequency of excitation light or differentfrequencies. It would be understood by those of ordinary skill in theart that different configurations are also possible. For instance,perimeter probe heads could be connected to a detector to detectfluorescence or different detectors and inner probe head could emitexcitation light. Alternatively, some combination of inner and perimeterprobe heads could emit excitation light and some combination of innerand perimeter probe heads could detect fluorescence.

While various embodiments/variations of the present invention have beendescribed above, it should be understood that they have been presentedby way of example only, and not limitation. Thus, the breadth and scopeof the present invention should not be limited by any of theabove-described exemplary embodiments. Further, unless stated, none ofthe above embodiments are mutually exclusive. Thus, the presentinvention may include any combinations and/or integrations of thefeatures of the various embodiments.

Additionally, while the processes described above and illustrated in thedrawings are shown as a sequence of steps, this was done solely for thesake of illustration. Accordingly, it is contemplated that some stepsmay be added, some steps may be omitted, and the order of the steps maybe re-arranged.

What is claimed is:
 1. A system for performing real-time PCR,comprising: a sample container for containing a sample of solutioncontaining real-time PCR reagents; a temperature control systemconfigured to cycle the temperature of the sample; an excitation sourcefor illuminating the sample; a fiber optic probe comprising: a bundle ofoptical fibers including a central optical fiber surrounded by aplurality of outer optical fibers, and a probe head connected to adistal end of the central optical fiber and positioned between thedistal end of the central optical fiber and the sample container forcontaining the sample, and a detector configured to detect emissionsexiting the proximal end of the central optical fiber, wherein theexcitation source is optically connected to each of said plurality ofouter optical fibers such that when the excitation source emitsexcitation light, the excitation light enters the outer optical fibersand then exits the outer optical fibers through a distal end of theoptical fibers.
 2. The system of claim 1, wherein the excitation sourcecomprises at least two light emitting devices, wherein each of the atleast two light emitting devices is optically connected to at lest oneof the outer optical fibers.
 3. The system of claim 1, wherein thesample container is in the form of a channel.
 4. The system of claim 1,wherein the probe head comprises a ball lens or a gradient index lens.5. The system of claim 1, wherein the diameter of the probe head is lessthan about 5 millimeters.
 6. The system of claim 5, wherein the diameterof the probe head is less than about 2 millimeters.